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A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species [EM-seq]

ABSTRACT: The derivation of authentic embryonic stem cells (ESCs) across mammalian species remains a major challenge. Here, we report the development of a defined, serum-free culture system, termed 6iL/E4, that enables the derivation and long-term self-renewal of ESCs across diverse mammalian species. To further characterize the state of 6iL-hiPSCs, we performed whole-genome bisulfite sequencing (WGBS) to compare 6iL-hiPSCs with hESCs cultured under mTeSR1 and t2iL/Go conditions. Library PCR amplification was performed for five cycles using NEBNext Multiplex Oligos for Enzymatic Methyl-seq. Equimolar amounts of each library were then pooled and quantified by qPCR before paired-end 150-bp sequencing on a 10B 300-cycle shared flow cell using the NovaSeq X Plus platform at the Advanced Genomics Core.

ORGANISM(S): Homo sapiens

PROVIDER: GSE333356 | GEO | 2026/06/02

REPOSITORIES: GEO

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A Universal 6iL/E4 Culture System for Deriving and Maintaining Embryonic Stem Cells Across Mammalian Species

Project description:The derivation of authentic embryonic stem cells (ESCs) across mammalian species remains a major challenge. Here, we report the development of a defined, serum-free culture system, termed 6iL/E4, that enables the derivation and long-term self-renewal of ESCs across diverse mammalian species. To further characterize the cellular states, we performed single-cell RNA sequencing (scRNA-seq) on 6iL-hiPSCs and 6iL-mESCs. Library preparation was carried out using the Single Cell 3′ RNA Prep T2 kit (Illumina). For indexing, the Mix 7 index set was used for 6iL-hiPSC samples and the Mix 8 index set for 6iL-mESC samples. Sequencing was performed on a NovaSeq X Plus platform using paired-end 150 bp reads (2 × 150 bp), generating approximately 200 million reads per sample (100 million reads per direction).

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